Murine Glioma Model Research Articles

Amplifying the Effects of Contrast Agents on Magnetic Resonance Images Using a Deep Learning Method Trained on Synthetic Data.

Artificial intelligence (AI) methods can be applied to enhance contrast in diagnostic images beyond that attainable with the standard doses of contrast agents (CAs) normally used in the clinic, thus potentially increasing diagnostic power and sensitivity. Deep learning-based AI relies on training data sets, which should be sufficiently large and diverse to effectively adjust network parameters, avoid biases, and enable generalization of the outcome. However, large sets of diagnostic images acquired at doses of CA outside the standard-of-care are not commonly available. Here, we propose a method to generate synthetic data sets to train an "AI agent" designed to amplify the effects of CAs in magnetic resonance (MR) images. The method was fine-tuned and validated in a preclinical study in a murine model of brain glioma, and extended to a large, retrospective clinical human data set. A physical model was applied to simulate different levels of MR contrast from a gadolinium-based CA. The simulated data were used to train a neural network that predicts image contrast at higher doses. A preclinical MR study at multiple CA doses in a rat model of glioma was performed to tune model parameters and to assess fidelity of the virtual contrast images against ground-truth MR and histological data. Two different scanners (3 T and 7 T, respectively) were used to assess the effects of field strength. The approach was then applied to a retrospective clinical study comprising 1990 examinations in patients affected by a variety of brain diseases, including glioma, multiple sclerosis, and metastatic cancer. Images were evaluated in terms of contrast-to-noise ratio and lesion-to-brain ratio, and qualitative scores. In the preclinical study, virtual double-dose images showed high degrees of similarity to experimental double-dose images for both peak signal-to-noise ratio and structural similarity index (29.49 dB and 0.914 dB at 7 T, respectively, and 31.32 dB and 0.942 dB at 3 T) and significant improvement over standard contrast dose (ie, 0.1 mmol Gd/kg) images at both field strengths. In the clinical study, contrast-to-noise ratio and lesion-to-brain ratio increased by an average 155% and 34% in virtual contrast images compared with standard-dose images. Blind scoring of AI-enhanced images by 2 neuroradiologists showed significantly better sensitivity to small brain lesions compared with standard-dose images (4.46/5 vs 3.51/5). Synthetic data generated by a physical model of contrast enhancement provided effective training for a deep learning model for contrast amplification. Contrast above that attainable at standard doses of gadolinium-based CA can be generated through this approach, with significant advantages in the detection of small low-enhancing brain lesions.

BIOL-17. DISSECTING THE EXPRESSION AND FUNCTION OF ANGIOPOIETIN-1 IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Angiopoietin-1 (Angpt1) is a secreted protein that promotes angiogenesis and vascular stability in development and certain disease states through activation of the Tie2 receptor. However, little is known about its expression or function within pediatric brain tumors. METHODS We utilized available datasets from patient samples and created murine models of pediatric high-grade glioma and ependymoma by in utero electroporation. Using a combination of transcriptomics and Angpt1-GFP reporter tumor models we evaluated Angpt1 expression across different types of pediatric brain tumors. We also applied inducible Angpt1 floxed mice to generate tumor-specific knock-out models. RESULTS Angpt1 is upregulated in glial brain tumors, including high-grade gliomas and ependymomas. We find Angpt1 is expressed by specific cell populations within gliomas and ependymomas, along with non-tumor reactive astrocytes and perivascular mural cells. Leveraging our in vivo brain tumor platform we have created tumor specific Angpt1 knock-out models by including a plasmid expressing Cre-recombinase. Ongoing studies aim to delineate the function of Angpt1 in tumor pathogenesis, including tumor angiogenesis and blood-brain barrier function. CONCLUSION Current work finds that Angpt1 is upregulated in glial brain tumors and nominates it as a potential modulator of brain tumor vascular biology, including tumor angiogenesis and permeability.

IMMU-21. ELUCIDATING THE CELLULAR MECHANISMS OF THERAPEUTIC BENEFIT UNDERLYING ADOPTIVE CELLULAR THERAPY IN MODELS OF GLIOMA

Abstract Among pediatric cancers, malignancies of the brain carry significant morbidity and mortality, harboring dismal prognosis and poor outcomes. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, including medulloblastoma and high-grade glioma. In this study, we characterized cell cycling differences of immune sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Five days following intracranial implantation of tumor cells, mice were treated with ACT which included the following elements: total body irradiation, hematopoietic stem cell transplantation, one dose of tumor-activated splenocytes and three weekly doses of dendritic cells that had been electroporated with tumor-derived ribonucleic acid. One week after the completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin-D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T-cells. In ACT-treated-mice, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME but reduced in spleen. These findings suggest that ACT alters immune cycling in peripheral lymphoid tissues and TME to potentially promote antitumor T-cell responses and minimize MDSC-mediated immunosuppression. While ACT-mediated alterations to immune cell cycling are likely a major contributor to the therapy’s benefit, it is unlikely to be the sole mechanism. Further exploration into other functional pathways, namely migration, is needed to explain changes to immune populations following ACT.

Efficacy and safety study of neoadjuvant efineptakin alfa (NT-I7) and pembrolizumab in recurrent glioblastoma.

TPS2085 Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor in adults. Despite maximal surgical resection, irradiation, and chemotherapy, median overall survival (OS) remains at only 30 weeks for recurrent GBM (rGBM) patients, emphasizing the need for novel treatments. GBM tumors are immunologically ‘cold,’ in that they have an immune-suppressive microenvironment with a low number of tumor-infiltrating lymphocytes (TIL). Interleukin 7 (IL-7) plays a key role in T cell development and survival. The physiological level of IL-7 increases to stimulate T cell expansion in lymphopenic condition. Previous studies have shown that lymphopenic GBM patients have shorter survival. However, their IL-7 levels are unexpectedly low. Efineptakin alfa (NT-I7), a long-acting IL-7, has been shown to increase effector CD8 T cells and reduce suppressor T-regulatory cells in murine GBM models, with subsequently improved OS. Further, we recently completed a Phase I study which showed that NT-I7 is safe, well- tolerated, and increased absolute lymphocyte counts (ALC) in newly diagnosed GBM patients. Phase II dosing was determined through this Phase I study reaching maximum tolerated dose. Thus, we hypothesized that NT-I7 may reverse GBM-associated immune suppression and potentiate the effect of an immune checkpoint inhibitor, leading to an increase in TILs and improved survival. Our preliminary data suggests that combining NT-I7 with pembrolizumab, an anti-PD-1 antibody, improves survival in murine glioma models. The purpose of this study is to evaluate the safety and efficacy of combining NT-I7 and pembrolizumab in rGBM patients. Methods: This is an open-label single arm one-stage Phase II study of NT-I7 and pembrolizumab as neoadjuvant and adjuvant therapy with surgery for adult patients with rGBM. We plan to enroll 30 evaluable patients, including a 6-patient safety run-in. Key eligibility criteria include candidates willing to undergo clinically indicated resection or biopsy, age ≥ 18 years, Karnofsky performance status ≥ 70, and dexamethasone dose > 2 mg/day ≤ 2 days prior to registration. The primary endpoint is a 9-month OS rate. Secondary endpoints include progression-free survival, objective response rate, changes in ALC, and adverse events profile. The correlative endpoints include studies to assess the anti-glioma immune response, changes in T cell subsets, T cell effector function, cytokine analysis, and immunohistochemical analysis of GBM tissue. Longitudinal cerebrospinal fluid proteomics is optional. This study opened on 1/20/2023. Two of the planned 30 patients had been enrolled by submission on 2/14/2023. Clinical trial information: NCT05465954 .

Reshaping the anti-tumor CD4 T-cell repertoire contributes to oncolytic virus therapeutic efficacy in malignant glioma

Abstract Malignant gliomas are deadly brain tumors with no cure. We engineered herpes simplex virus (HSV)-1 and developed oncolytic HSV (oHSV) to treat brain tumors, given its potential to selectively kill tumor cells while sparing normal brain cells, and to boost anti-tumor immunity. The therapy shows great promise based on our Phase I trial results. However, the precise mechanisms for its enhanced anti-tumor immune responses remain poorly understood. Using an oHSV secreting murine IL-12 (M002), murine intracranial glioma models and adoptive transfer approaches, we observed that M002 treatment induced unique CD4 +T-cell populations to acquire cytolytic effector phenotypes, promoting anti-glioma responses in an MHCII-dependent manner. Notably, single-cell RNA sequencing and T-cell receptor (TCR) repertoire analysis revealed that M002 treatment shifted the TCR dominance while reducing the clonal diversity. These results suggest that the capacity of oncolytic virus therapy to reshape the anti-tumor CD4 +T-cell repertoire contributes to its improved efficacy. Insights obtained from this study can facilitate the development of better therapeutic strategies for brain tumor patients. Supported by DoD W81XH-18-1-0315 and UAB Faculty Start-up Funds (to JWL)

Elucidating the cellular mechanisms underlying adoptive cell transfer in models of glioma

Abstract Despite advances in solid tumor biology and oncology, pediatric and adult primary brain cancer and metastasis to the brain harbor dismal prognosis and poor outcomes. One avenue to overcome these clinical challenges is through developing robust immunotherapy to better target the tumor. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, namely medulloblastoma, brain stem glioma and glioblastoma. In this study, we characterized cell cycling differences of immune cellular sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Following completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T cells. In mice receiving ACT, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME, but reduced in spleen. These findings suggest that ACT changes immune cycling and migration dynamics in lymphoid tissue and TME to potentially promote antitumor T-cell responses and minimize tumor-mediated immunosuppression. 5R01NS112315-04

378 RAGE Ablation Attenuates Glioma Progression and Enhances Tumor Immune Responses by Suppressing Galectin-3 Expression

INTRODUCTION: Malignant gliomas consist of heterogenous cellular components that have adopted multiple overlapping escape mechanisms that overcome both targeted and immune-based therapies. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that is activated by diverse proinflammatory ligands present in the tumor microenvironment (TME). Activation of RAGE by its ligands stimulates multiple signaling pathways that are important in tumor growth, invasion, and immune escape. METHODS: We evaluated genetic ablation of RAGE on the tumorigenicity of two syngeneic murine glioma models. RAGE expression was inhibited in the GL261 and K-Luc gliomas by shRNA and CRSPR/Cas9 techniques prior to intracranial implantation. Tumor growth, invasion, and inflammatory responses were examined by immunohistochemistry, qPCR, ELISA, western blotting, NanoString, flow cytometry, and in vivo survival analysis. RESULTS: Intracellular RAGE ablation abrogated glioma growth and invasion by suppressing AKT and ERK1/2 activities and by downregulating MMP9 expression. Interestingly, RAGE inhibition also enhanced tumor inflammatory responses by downregulating the expression of galectin-3, rendered the immunotherapy resistant K-Luc gliomas responsive to immune checkpoint blockade therapy, and significantly increased survival. CONCLUSIONS: This study is the first to link RAGE expression with galactin-3 as an immune modulator in glioblastoma and revealed that the effect of glioma RAGE expression on TME was mediated through downregulation of Gal-3 and subsequent polarization of Tumor-associated macrophages. The therapeutic significance of RAGE downregulation was demonstrated by sensitizing a highly invasive and immune-resistant glioma model to immune checkpoint blockade and strongly supports the development of RAGE ablation as a complementary treatment strategy for malignant gliomas.

Textural Features of Mouse Glioma Models Measured by Dynamic Contrast-Enhanced MR Images with 3D Isotropic Resolution.

This paper investigates the effect of anisotropic resolution on the image textural features of pharmacokinetic (PK) parameters of a murine glioma model using dynamic contrast-enhanced (DCE) MR images acquired with an isotropic resolution at 7T with pre-contrast T1 mapping. The PK parameter maps of whole tumors at isotropic resolution were generated using the two-compartment exchange model combined with the three-site-two-exchange model. The textural features of these isotropic images were compared with those of simulated, thick-slice, anisotropic images to assess the influence of anisotropic voxel resolution on the textural features of tumors. The isotropic images and parameter maps captured distributions of high pixel intensity that were absent in the corresponding anisotropic images with thick slices. A significant difference was observed in 33% of the histogram and textural features extracted from anisotropic images and parameter maps, compared to those extracted from corresponding isotropic images. Anisotropic images in different orthogonal orientations demonstrated 42.1% of the histogram and textural features to be significantly different from those of isotropic images. This study demonstrates that the anisotropy of voxel resolution needs to be carefully considered when comparing the textual features of tumor PK parameters and contrast-enhanced images.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ Tcells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Mathematical modelling of the dynamics of image-informed tumor habitats in a murine model of glioma

Tumors exhibit high molecular, phenotypic, and physiological heterogeneity. In this effort, we employ quantitative magnetic resonance imaging (MRI) data to capture this heterogeneity through imaging-based subregions or “habitats” in a murine model of glioma. We then demonstrate the ability to model and predict the growth of the habitats using coupled ordinary differential equations (ODEs) in the presence and absence of radiotherapy. Female Wistar rats (N = 21) were inoculated intracranially with 106 C6 glioma cells, a subset of which received 20 Gy (N = 5) or 40 Gy (N = 8) of radiation. All rats underwent diffusion-weighted and dynamic contrast-enhanced MRI at up to seven time points. All MRI data at each visit were subsequently clustered using k-means to identify physiological tumor habitats. A family of four models consisting of three coupled ODEs were developed and calibrated to the habitat time series of control and treated rats and evaluated for predictive capability. The Akaike Information Criterion was used for model selection, and the normalized sum-of-square-error (SSE) was used to evaluate goodness-of-fit in model calibration and prediction. Three tumor habitats with significantly different imaging data characteristics (p < 0.05) were identified: high-vascularity high-cellularity, low-vascularity high-cellularity, and low-vascularity low-cellularity. Model selection resulted in a five-parameter model whose predictions of habitat dynamics yielded SSEs that were similar to the SSEs from the calibrated model. It is thus feasible to mathematically describe habitat dynamics in a preclinical model of glioma using biology-based ODEs, showing promise for forecasting heterogeneous tumor behavior.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

111In and 131I labeled nimotuzumabs for targeted radiotherapy of a murine model of glioma

111In and 131I labeled nimotuzumabs for targeted radiotherapy of a murine model of glioma

Wnt signaling regulates MFSD2A-dependent drug delivery through endothelial transcytosis in glioma.

Systemic delivery of anti-tumor therapeutic agents to brain tumors is thwarted by the blood-brain barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). A failure of pharmacological compounds to cross BBB is one culprit for the dismal prognosis of glioblastoma (GBM) patients. Identification of novel vascular targets to overcome the challenges posed by the BBB in tumors for GBM treatment is urgently needed. Temozolomide (TMZ) delivery was investigated in CT2A and PDGFB-driven RCAS/tv-a orthotopic glioma models. Transcriptome analysis was performed on ECs from murine gliomas. Mfsd2a deficient, Cav1 deficient, and Mfsd2a EC-specific inducible mice were developed to study the underlying molecular mechanisms. We demonstrated that inhibiting Wnt signaling by LGK974 could increase TMZ delivery and sensitize glioma to chemotherapy in both murine glioma models. Transcriptome analysis of ECs from murine gliomas revealed that Wnt signaling inhibition enhanced vascular transcytosis as indicated by the upregulation of PLVAP and downregulation of MFSD2A. Mfsd2a deficiency in mice enhances TMZ delivery in tumors, whereas constitutive expression of Mfsd2a in ECs suppresses the enhanced TMZ delivery induced by Wnt pathway inhibition in murine glioma. In addition, Wnt signaling inhibition enhanced caveolin-1 (Cav1)-positive caveolae-mediated transcytosis in tumor ECs. Moreover, Wnt signaling inhibitor or Mfsd2a deficiency fails to enhance TMZ penetration in tumors from Cav1-deficient mice. These results demonstrated that Wnt signaling regulates MFSD2A-dependent TMZ delivery through a caveolae-mediated EC transcytosis pathway. Our findings identify Wnt signaling as a promising therapeutic target to improve drug delivery for GBM treatment.

Differential roles of type I interferon signaling in tumor versus host cells in experimental glioma models

Despite multimodal treatment approaches including surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma remains in the range of one year and thus poor. Type I interferons (IFN) are involved in immune responses to viral infection and exhibit anti-tumor activity in certain cancers.Here we explored the biological relevance of constitutive type I IFN signaling in murine glioma models in vitro and in vivo. CT-2A, GL-261, SMA-497, SMA-540 and SMA-560 murine glioma cells expressed IFN type I receptors IFNAR1 and IFNAR2 and were responsive to exogenous IFN stimulation. CRISPR/Cas9-mediated deletion of IFNAR1 decreased the baseline expression of type I IFN response genes in GL-261 cells, but neither in CT-2A nor in SMA-560 cells. IFNAR1 deletion slowed growth in GL-261 and SMA-560, but not in CT-2A cells. However, only the growth of IFNAR1-depleted GL-261 tumors and not that of SMA-560 tumors was delayed in vivo upon orthotopic tumor cell implantation into syngeneic mice. This survival gain was no longer detected when the IFNAR1-depleted GL-261 cells were inoculated into IFNAR1-deficient mice. Altogether these data suggest that constitutive type I IFN signaling in gliomas may be pro-tumorigenic, but only in a microenvironment that is proficient for type I IFN signaling in the host.

RAGE ablation attenuates glioma progression and enhances tumor immune responses by suppressing galectin-3 expression.

Malignant gliomas consist of heterogeneous cellular components that have adopted multiple overlapping escape mechanisms that overcome both targeted and immune-based therapies. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that is activated by diverse proinflammatory ligands present in the tumor microenvironment. Activation of RAGE by its ligands stimulates multiple signaling pathways that are important in tumor growth and invasion. However, treatment strategies that only target the interaction of RAGE with its ligands are ineffective as cancer therapies due to the abundance and diversity of exogenous RAGE ligands in gliomas. As an alternative approach to RAGE ligand inhibition, we evaluated the genetic ablation of RAGE on the tumorigenicity of 2 syngeneic murine glioma models. RAGE expression was inhibited in the GL261 and K-Luc gliomas by shRNA and CRSPR/Cas9 techniques prior to intracranial implantation. Tumor growth, invasion, and inflammatory responses were examined by histology, survival, Nanostring, and flow cytometry. Intracellular RAGE ablation abrogated glioma growth and invasion by suppressing AKT and ERK1/2 activities and by downregulating MMP9 expression. Interestingly, RAGE inhibition in both glioma models enhanced tumor inflammatory responses by downregulating the expression of galectin-3 and potentiated immunotherapy responses to immune checkpoint blockade. We demonstrated that intracellular RAGE ablation suppresses multiple cellular pathways that are important in glioma progression, invasion, and immune escape. These findings strongly support the development of RAGE ablation as a treatment strategy for malignant gliomas.

TMIC-42. LEVERAGING B CELL IMMUNITY TO PROMOTE IMMUNOTHERAPY IN GLIOBLASTOMA

Abstract Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in melanoma and sarcoma, amongst other models, have revealed the accumulation of germinal-center-like B cells as a key survival predictor post-PD1 blockade. We seek to leverage B cell immunity to enhance immunotherapy effectiveness in glioblastoma (GBM). In human GBM and murine glioma models, we found that B cells in the tumor microenvironment (TME) are activated, but the expression of co-inhibitory molecules such as CD32 and CD72 blocks downstream effector function. Transcriptomic analysis showed high expression of inhibitory TGFβ receptors on B cells and high levels of TGFβ1 cytokine in the TME. We showed direct inhibition of B cell function through TGFβ signaling that could be prevented with TGFβ receptor blockade. Spatial multiplex immunofluorescence analysis of the TME revealed that tumor and myeloid cells express high levels of TGFβ and are also near B cells, allowing for TGFβ-mediated B cell inhibition. Blocking the TGFβ pathway via transgenic mice with TGFβ receptor knockout on B cells or TGFβ cytokine knockouts in myeloid cells, or generation of a CT2A tumor line with TGFβ cytokine knockdown, all demonstrated a survival benefit and more germinal-center-like B cells. There was also increased T cell proliferation and anti-tumor cytotoxicity. Finally, inhibiting αVβ8 integrin, a required factor that releases active TGFβ, is a translatable approach that also increased B cell proliferation and animal survival. Dual treatment with αVβ8+PD1 blockade showed the most potent survival as well as immunological memory against tumor re-challenge. Analysis of the B and T cell compartments after dual treatment showed synergy, with robust cellular proliferation and functional differentiation of plasmablasts and effector T cells. Collectively, our study highlights the importance of B cells in the TME and a remodeled approach to boost the effects of immunotherapy against GBM.

IMMU-20. MODULATION OF THE PERIPHERAL T CELL BIOLOGY IN GLIOBLASTOMA

Abstract Cancer is a systemic disease. Due to the exceedingly rare occurrence of metastasis of cerebral glioma, systemic alterations have, however, not been considered to play a major role in disease progression of glioma. T cells orchestrate the adaptive immune response in an antigen-specific, cytokine mediated manner. The aim of our study was to investigate how cerebral glioma impacts systemic T cell immunobiology. We performed gene expression profiling of peripheral blood T cells in patients with IDHwt glioblastomas as well as in a murine glioma model. In addition, we analyzed the levels of soluble immune biomarkers in patient blood and performed flow-cytometric phenotyping of human peripheral blood CD3+ T cells. We discovered a significant skewing of peripheral T cell phenotypes in IDHwt glioblastoma patients compared to healthy donors (HD), showing CD4+ TH1 expansion and reduced numbers of T follicular helper cells (TFH), TH1* and mucosa associated invariant T (MAIT) cells, while TH2 and TH17 percentages remained stable. Interestingly, peripheral memory CD4+ T cells exhibited reduced Fas and PD-1 expression, while CD8 T cells were primarily affected in the non-memory CD45RA+ compartment, displaying reduced numbers of CD8+ CD127+ CD27+ cells. Compared to healthy individuals, GBM patients had significantly increased levels of soluble CD27 while levels of soluble CD25 were reduced (p &amp;lt; 0.05). GSEA and ORA analysis of differentially expressed genes in murine gliomas showed alterations in RNA binding and processing, as well as ribosomal activity in both cell types, indicating systemic modulation in translational- and cell cycle pathways in glioblastoma. Taken together, our results demonstrate a significant skewing of the peripheral T immunobiology in patients with IDHwt gliomas. Our data highlights the importance of considering malignant glioma as a systemic disease that fundamentally alters the immune repertoire in affected patients.

NIMG-41. NON-INVASIVE TRACKING OF T-CELL RECRUITMENT TO THE TUMOR MICROENVIRONMENT IN A MURINE GLIOMA MODEL BY HIGH FIELD CELLULAR MRI

Abstract Gliomas are characterized by increased T cell exhaustion and poor T cell infiltration into the tumor as well as an overall highly immunosuppressive tumor microenvironment (TME). Response rates in preclinical glioma models and patients to promising new therapeutic approaches in the field of immunotherapies - such as checkpoint blockade, vaccines and adoptive therapy with chimeric antigen receptor (CAR) or T cell receptor (TCR)-transgenic T cells - remain heterogeneous. This demonstrates the need for non-invasive tracking of T cell recruitment to the TME in order to monitor T cell activating immunotherapies, adapt therapeutic strategies and predict treatment outcome. Iron oxide nanoparticles (NP) can be visualized non-invasively by magnetic resonance imaging (MRI) and dedicated MRI sequences such as T2* mapping. Using isolated murine T cells cultures we show that labeling of T cells with iron oxide NP as contrast agent is feasible and does not impact T cell viability and functionality as assessed by cytokine secretion and antigen-specific killing activity in vitro. We demonstrate that adoptively transferred T cells can be visualized intratumorally in a murine glioma model by high field MRI at 9.4 Tesla with high sensitivity and that T cells can be tracked non-invasively in a time course of over one week. Ongoing work assesses preclinical efficacy of adoptive T cell therapy targeting well characterized model antigens expressed in experimental gliomas using longitudinal MRI to visualize spatial and temporal T-cell dynamics in the TME. Correlative methods include immunohistochemistry, flow cytometry, tissue clearing and ultramicroscopy. We hypothesize that T cell distribution and numbers may predict therapeutic efficacy and correlate with treatment outcome in experimental gliomas.

CTIM-23. DOSE ESCALATION TRIAL OF FC-ENGINEERED ANTI-CD40 MONOCLONAL ANTIBODY (2141-V11) ADMINISTERED INTRATUMORALLY WITH D2C7-IT VIA CONVECTION-ENHANCED DELIVERY (CED) FOR RECURRENT MALIGNANT GLIOMAS (RMGS)

Abstract BACKGROUND D2C7-IT, a novel immunotoxin-based cytotoxic therapy, targets epidermal growth factor receptor (EGFR) and mutant EGFR variant III. In preclinical studies, D2C7-IT kills tumor cells and prolongs survival, but is unable to generate cures in all animals. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. Eliminating glioblastoma immunosuppression via CD40 co-stimulation is anticipated to enhance D2C7-IT-induced antitumor responses. In murine glioma models, CED of D2C7-IT+αCD40 generated cures and long-term tumor-specific adaptive immunity. Hence, we are conducting a phase 1 trial of D2C7-IT+2141-V11 (αhuman-CD40) administered via CED in rMG patients. METHODS Eligibility includes adult patients with solitary supratentorial rMG (WHO grade 3/4); ≥ 4 weeks after chemotherapy, bevacizumab, or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing doses of 2141-V11 to determine the maximum tolerated dose when administered sequentially following D2C7-IT (166,075 ng) via CED at 0.5 mL/hr. Five dose levels (DLs) are evaluated (2141-V11 at: #1: 0.70 mg; #2: 2.0 mg; #2**: 3.0 mg #2*: 4.0 mg; #3: 7.0 mg). RESULTS As of May 29, 2022, 13 patients were treated (3 patients on DL1, DL2, and DL2**; 2 patients on DL3 and DL2*). No dose-limiting toxicities were observed; however, lower DLs were added due to higher frequency of adverse events (AEs) expected with D2C7-IT+2141-V11 with DL3 and DL2* (fever, neurologic symptoms). All patients remain alive 0.7-10.6 months after therapy. Grade ≥ 2 AEs due to D2C7-IT+2141-V11 include: headache (grade 3, n = 1; grade 2, n = 4); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1); dysphasia (grade 3, n = 1); seizures (grade 2; n = 2); fever (grade 2; n = 2); and one each of grade 2 depressed level of consciousness, fatigue, and concentration impairment. Enrollment is ongoing. CONCLUSIONS Intratumoral administration of D2C7-IT+2141-V11 via CED is safe, encouraging efficacy results are observed.